David biweekly meeting
project updates
Project 4: Reconstitution of endocytic actin network
recent progress
- Danielle had to delay sectioning samples due to covid in her family; should be resuming this week
next steps
- EM imaging when sectioning/staining is done
Project 5: Experimental role of phase-separated protein droplet in endocytosis
recent progress
eps15 appears to express well in e. coli in test expression (replicating Stachowiak lab paper)
- green ladder band is ~100kD, same as eps15
- lanes: 0,2,4,6,20,22,24,26,28 hr induction
next steps
- see plan for Nikhil's protein purification project
brainstorming on how to adapt this system for complementing the model
- Tyler suggested finding a substrate with holes approximately the size of the nanoparticles in order to achieve lower curvature
- try making cytoplasmic extract from ede1 overexpression strain
- already tried the 3x adaptor deletion strain; did not produce condensates
Project 7: Mechanical regulation of CME by protein LLPS
recent progress
- with Tyler's help, identified a term that was missing from my model that caused the simulations I showed last time to not run correctly
next steps
- add in the missing term
- run a simulation of successful endocytosis mediated by the coat bending energy
- build final version of model with protein condensate
plan for Nikhil's protein purification project
- express and purify His-tagged GFP in E. coli
- insert GFP into His6-eps15 plasmid from Stachowiak lab
- express and purify His6-eps15-GFP from E. coli
- purify His6-eps15
- purify Fcho1
- reproduce phase separation experiments